Masahide Noji

Effect of zeta potential of cationic liposomes containing cationic cholesterol derivatives on gene transfection

Cationic liposomes are known to be useful tools for gene transfection. However, the relation between transfection efficiency and physicochemical properties of liposomes has not been well understood. Here, we synthesized eight cationic derivatives of cholesterol which contain a tertiary amino head group with a different spacer arm. Transfection of plasmid pSV2CAT DNA into cells was done by cationic liposomes made of a mixture of dioleoylphosphatidylethanolamine (DOPE) and each cationic cholesterol derivative. At the same time we measured zeta potential of cationic liposomes by laser Doppler spectroscopy. The present results indicated that zeta potentials of cationic liposomes were well related to transfection activity of pSV2CAT DNA. This suggested that zeta potential of cationic liposomes is one of important factors which control gene transfection.

The crystal structures and absolute configurations of the anti-tumor complexes Pt(oxalato)(1R,2R-cyclohexanediamine) and Pt(malonato)(1R,2R-cyclohexanediamine)

Abstract The absolute configurations of the anti-tumor complexes [Pt(oxalato)(trans-l-dach)] and [Pt(malonato) (trans-l-dach)] (trans-l-dach = 1R,2R-cyclohexanediamine) have been determined by X-ray anomalous scattering techniques. These complexes are particularly interesting because they show higher anti-tumor activity than the corresponding Pt complexes with other 1,2-cyclohexanediamine(dach) ligands, namely those with trans-d-dach (1S,2S-dach) or cis-dach (1R,2S-dach). The oxalato and malonato ligands are found to bind to the Pt atom in a chelating fashion, through one oxygen atom from each of the two carboxylate groups. Crystallographic details: Pt(oxalato)(trans-l-dach): space group P2 1 (monoclinic); a = 11.230(11) A, b = 9.914(5) A, c = 4.716(3) A, β = 90.86(6)°; R = 4.0% for 1256 reflections. Pt(malonato)(trans-l-dach): space group P2 1 (monoclinic); a = 11.568(5) A, b = 10.007(5) A, c = 5.187(3) A, β = 99.16(4)°; R = 4.8% for 1675 reflections.

Cationic cholesterol with a hydroxyethylamino head group promotes significantly liposome-mediated gene transfection

A novel cationic cholesterol derivative with a hydroxyethylamino head group, cholesteryl‐3β‐carboxyamidoethylene‐N‐hydroxyethylamine (II), has been synthesized and used for liposome‐mediated gene transfection. The cationic liposomes containing the derivative (II) facilitated greatly pSV2CAT gene transfection into mouse NIH3T3 and L929 cells in the absence of serum. The transfection efficiency was much higher than those by the cationic liposomes containing cationic derivatives with a dialkylamino head group (I, III or IV). Further, the efficiency by the cationic liposomes with the derivative (II) was not so much decreased in the presence of serum. This suggested that a novel cationic cholesterol derivative (II) should be very promising in liposome‐mediated gene transfection of plasmid and antisense DNA into target cells.

An orally active antitumor cyclohexanediamine-Pt(IV) complex: trans,cis,cis-bis(n-valerato) (oxalato)(1R,2R-cyclohexanediamine)Pt(IV)

In order to develop orally active antitumor platinum complexes, several cyclohexanedlamine-Pt(IV) complexes of a general formula trans,cis,cis-[Pt(IV) (OCOCnHn+1)2 (oxalato)(1R,2R-cyclohexanediamine)] were synthesized by derivatizing oxaliplatin [Pt(III)(oxalato)(1R,2R-cyclohexanediamine), I-OHP], which is a potent antitumor cyclohexanedlamine-Pt(II) complex we have prepared and now undergoing clinical trials. The I-OHP derivatives were found to be stable, lipophilic and reduced to yield I-OHP, an active species, quantitatively by ascorbate in vitro. All the derivatives were antitumor active against mouse lymphocytic leukemia L1210 when given i.p. In particular, trans-bis-valerato-oxalato-1R,2R-dach-Pt(IV), C5-OHP, showed markedly high activity. C5-OHP also exhibited significant antitumor activity against L1210 when orally administered. C5-OHP was considered to be a suitable candidate for the oral cancer chemotherapy agent to be developed.

A fluorescent molecular rotor probes the kinetic process of degranulation of mast cells

A confocal fluorescence microscope was used to study the exocytotic secretory processes of mast cells in combination with an fluorescent molecular rotor, 9-(dicyanovinyl)julolidine (DCVJ). DCVJ is known to be an unique fluorescent dye which increases its quantum yield with decreasing intramolecular rotation. Here, DCVJ-loaded peritoneal rat mast cells were stimulated with compound 48/80 and their fluorescence images were compared with fluorescence calcium images of fluo-3-loaded mast cells. Subsequent to transient increases in intracellular free calcium ion concentration, DCVJ fluorescence increased dramatically in the cytoplasm and formed a ring-like structure around the nucleus, suggesting the possibility that the dye bound to the proteins composing the cytoskeletal architecture. Furthermore, the increases of DCVJ fluorescence intensities were mostly blocked in the presence of cytochalasin D (10 microM). However, fluo-3 fluorescence intensities still increased after addition of compound 48/80.

Coordination chemical studies on metalloenzymes: Measurement of binding constant between apo-tyrosinase and copper ion

Abstract The behavior of complexing agents for the copper removal reaction was studied by the equilibrium dialysis method. In the copper removal reaction, complexing agents are divided into two types: those that are reducing agents and those that are not. Sodium cyanide and sodium thiosulfate are of the first type, and 8-hydroxyquinoline-5-sulfonic acid, 2,2′-bipyridyl, and picolinic acid are of the second type. From equilibrium dialysis with the first type of complexing agent, the apparent binding constant (pH 6.0) between cuprous ions and apotyrosinase was calculated to be 10 15 m −1 . Similarly, the apparent binding constant (pH 6.0) between cupric ions and apo-tyrosinase was about 10 13 m −1 , which was calculated from equilibrium dialysis with the second type of complexing agent. The apparent binding constant between cuprous ions and apo-tyrosinase was larger than that between cupric ions and apo-tyrosinase.

Orally Active DACH-Pt (IV) Compounds

Since the discovery of the antitumor activity of cisplatin by Rosenberg, et al.1 in 1969, many attempts have been made to prepare highly antitumor active Pt complexes without severe toxicities. The authors discovered a simple preparative separation method of 1,2-diaminocyclohexane (dach) geometrical isomers, cis and trans2, by Nickel complex formation and trans isomers were resolved into d and l optical isomers by the conventional method.

Antitumor activity of steroid-containing platinum (II) complexes of 1R,2R-cyclohexanediamine and 2-(aminomethyl)-cyclohexylamine isomers against leukemia L1210

Various steroid-containing platinum (Pt) complexes of 1R,2R-cyclohexanediamine (= 1R,2R-dach) and cis-dl- and trans-dl-2-(aminomethyl)cyclohexylamine (= amcha) were synthesized and their antitumor activity was screened against leukemia L1210 according to the Pt Analog Study Protocol. Among the Pt complexes prepared, Pt 1R,2R-dach complexes of cortisone, hydrocortisone, methylprednisolone, testosterone, estriol and progesterone showed very high antitumor activity. Pt complexes of cis-dl- and trans-dl-amcha prednisolone, Pt(17-OH-progesterone) (trans-dl-amcha), Pt complexes of cis-dl- and trans-dl-amcha progesterone were found to be very effective.

Developmental Approach to Prepare New Types of Antitumor Platinum Complexes with Dual Function

cis-Platin, discovered by B. Rosenberg, et al. in 1969 is nowadays one of the best and widely used antitumor agents in cancer chemotherapy of testicular, ovarian, bladder cancers, as well as head and neck cancer. Various attempts have been made to prepare future generation antitumor Pt complexes, ameliorating severe toxicities. Carboplatin has been developed as the 2nd generation Pt complex of less nephrotoxic agent, but it still shows severe myelosuppression and cross-resistance.

Antibodies against (1R,2R)-cyclohexanediamineplatinum(II)-DNA adduct recognize the conformational differences of isomeric analogues of cyclohexanediamine

Antibodies reactive to (1R,2R)-cyclohexanediamineplatinum(II)-DNA ((1R,2R)-cyclohexanediamine: 1R,2R-dach) adducts were elicited by immunization of rabbit with calf thymus DNA modified by Pt(1R,2R-dach)Cl2 at a ratio of bound platinum per nucleotide ((D/N)b) of 0.0335. In an enzyme-linked immunosorbent assay (ELISA), the binding of specific antibodies to Pt(1R,2R-dach)-DNA adduct (60 microliters of 1.235 x 10(-7) M Pt in each wells) on the assay plate was competitively inhibited by Pt(1R,2R-dach)-DNA adduct ((D/N)b = 0.0653) in the solution. Almost equal inhibition was observed with Pt(1S,2S-dach)-DNA ((D/N)b = 0.0412), an optical isomer of 1R,2R-dach. Pt(1R,2S-dach)-DNA ((D/N)b = 0.0371) and Pt(1R,3S-dach)-DNA ((D/N)b = 0.0281) in which the cyclohexane ring is stereochemically perpendicular to the platinum chelate plane, also inhibited antibody binding, but these adducts gave only incomplete inhibition at higher Pt-DNA adduct concentrations. Although Pt(1R,2R-dach)-d(GpG) and Pt(1R,2R-dach)(NH3)2 inhibited antibody binding, the affinity of the antibody for Pt(1R,2R-dach)(NH3)2 was lower than with Pt(1R,2R-dach)-DNA, and the inhibition behavior of Pt(1R,2R-dach)-d(GpG) was biphasic, i.e., at the lower concentration the inhibition curve was consistent with that of Pt(1R,2R-dach)-DNA, but at the higher concentration it shifted to that of Pt(1R,2R-dach)(NH3)2. The affinity of the antibody for cis-DDP was markedly lower than with Pt(1R,2R-dach)(NH3)2. These facts suggest that the antibodies may bind to the substituents (the platinum and its surroundings) of the various Pt complexes rather than the DNA structure altered by platinum binding.